Project Summary The ferret, Mustela putorius furo, is the gold-standard animal model for studying the biology and pathogenesis of influenza viruses. This small animal model has also been utilized to study the biology and pathogenesis of other human respiratory viruses including respiratory syncytial virus, metapneumovirus, measles virus, mumps virus, parainfluenza viruses, severe acute respiratory syndrome coronavirus (SARS- CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). Ferret models of cystic fibrosis and cigarette smoke-induced chronic obstructive pulmonary disease (COPD) have also been developed. The ferret and human respiratory tracts share anatomic and physiological similarities, including: elongated trachea; similar distribution of sialic acids which are receptors for some respiratory viruses; and similar pulmonary physiology. The ferret model is amenable to non-terminal collection of nasal wash samples, oropharyngeal swabs, and bronchoalveolar lavage fluid (BALF) which allows assessment of immune responses and virus replication at these respiratory mucosal sites. The average size and total blood volume of a ferret allows for longitudinal assessment of serum and peripheral blood mononuclear cell responses. Thus, the ferret model is amenable to the longitudinal collection of the same types of clinical samples collected from humans. The ferret is an invaluable small animal model to investigate the pathobiology of respiratory tract disease resulting from virus infection, and to study the efficacy of prophylactic vaccines and antiviral compounds to prevent disease. However, the lack of an extensive repository of immunological reagents such as those available for mouse or human studies is a common criticism of the ferret model. The significance of this project is that it addresses this lack of immunological reagents, which is perceived as a critical barrier limiting the utility of the ferret model, by developing novel monoclonal antibodies that are essential to establish new assays to quantify B and T cell responses of ferrets. The overall goal of this project is to investigate the mucosal immune responses of the ferret respiratory tract to improve the predictability and reproducibility of ferret studies that model the immune responses of the human respiratory tract to infectious diseases or mucosal immunization. Toward this goal, we propose to clone up to ten genes encoding specific ferret B or T cell markers, and then develop corresponding recombinant proteins that will be used to immunize mice for the production of monoclonal antibodies. Our newly developed monoclonal antibodies specific for ferret B and T cells will be complemented with already commercially available immunological reagents to establish flow cytometry assays and enzyme-linked immunosorbent assays (ELISA) that will enable quantitative studies of the systemic and mucosal immune responses of the ferret respiratory tract to infection or vaccination. We propose to use the immunological reagents developed to examine the hypothesis that autophagy can be manipulated to influence the quality of the CD4+ T cell-dependent antibody responses to influenza vaccination.